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An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIV mac 239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection., Competing Interests: Competing interests Authors declare that they have no competing interests.

Antimicrobial resistance (AMR) represents a growing public health threat that arises at the interface between animal, human, and environmental health. Although the pathways promoting the development of AMR are well characterized in human health settings, data within the veterinary medical world are less abundant, particularly from fields focusing on nontraditional species, such as nonhuman primates (NHPs). The purpose of this study was to describe trends in sample submission for bacterial culture, characterize patterns of microbial growth and any changes in AMR and susceptibility over time, and inform best practices for veterinary antimicrobial stewardship in a captively-housed, indoor NHP colony. Electronic health records from the Wisconsin National Primate Research Center were analyzed across a 10-y period using SAS Studio. There was an increasing pattern of sample submissions for culture and susceptibility analyses, with no corresponding increases in resistance to relevant antibiotics for potential zoonotic pathogens, such as Escherichia coli or Shigella species. Trends are suggestive of appropriate antimicrobial stewardship practices that were responsive to the medical needs of Wisconsin National Primate Research Center animals, as well as the needs of the larger research community at the University of Wisconsin-Madison. These findings can inform veterinary professionals working with NHPs and contribute to the growing body of literature surrounding AMR in nontraditional species.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell-derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect, aiming to support ventricular function. Here, we report successful engraftment of human induced pluripotent stem cell-derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 10 6 cells/kg) and high-dose (10 × 10 6 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells, with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The common marmoset ( Callithrix jacchus ) is one of the most widely used nonhuman primate models of human disease. Owing to limitations in sequencing technology, early genome assemblies of this species using short-read sequencing suffered from gaps. In addition, the genetic diversity of the species has not yet been adequately explored. Using long-read genome sequencing and expert annotation, we generated a high-quality genome resource creating a 2.898 Gb marmoset genome in which most of the euchromatin portion is assembled contiguously (contig N50 = 25.23 Mbp, scaffold N50 = 98.2 Mbp). We then performed whole genome sequencing on 84 marmosets sampling the genetic diversity from several marmoset research centers. We identified a total of 19.1 million single nucleotide variants (SNVs), of which 11.9 million can be reliably mapped to orthologous locations in the human genome. We also observed 2.8 million small insertion/deletion variants. This dataset includes an average of 5.4 million SNVs per marmoset individual and a total of 74,088 missense variants in protein-coding genes. Of the 4956 variants orthologous to human ClinVar SNVs (present in the same annotated gene and with the same functional consequence in marmoset and human), 27 have a clinical significance of pathogenic and/or likely pathogenic. This important marmoset genomic resource will help guide genetic analyses of natural variation, the discovery of spontaneous functional variation relevant to human disease models, and the development of genetically engineered marmoset disease models.

Recent human decedent model studies 1,2 and compassionate xenograft use 3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts., (© 2023. The Author(s).)

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques ( Macaca fascicularis ) inoculated with ZIKV or SPONV were susceptible to ZIKV but resistant to SPONV infection. In contrast, rhesus macaques ( Macaca mulatta ) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis and suggest that the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV.

Barbiturates and benzodiazepines are GABA A -receptor agonists and potent antiseizure medications. We reported that exposure of neonatal macaques to combination of phenobarbital and midazolam (Pb/M) for 24 h, at clinically relevant doses and plasma levels, causes widespread apoptosis affecting neurons and oligodendrocytes. Notably, the extent of injury was markedly more severe compared to shorter (8 h) exposure to these drugs. We also reported that, in the infant macaque, mild hypothermia ameliorates the apoptosis response to the anesthetic sevoflurane. These findings prompted us explore whether mild hypothermia might protect infant nonhuman primates from neuro- and gliotoxicity of Pb/M. Since human infants with seizures may receive combinations of benzodiazepines and barbiturates for days, we opted for 24 h treatment with Pb/M. Neonatal rhesus monkeys received phenobarbital intravenously, followed by midazolam infusion over 24 h under normothermia (T > 36.5 °C-37.5 °C; n = 4) or mild hypothermia (T = 35 °C-36.5 °C; n = 5). Medication doses and blood levels measured were comparable to those in human infants. Animals were euthanized at 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated. Extensive degeneration of neurons and oligodendrocytes was seen at 36 h in both groups within neocortex, basal ganglia, hippocampus and brainstem. Mild hypothermia over 36 h (maintained until terminal perfusion) conferred no protection against the neurotoxic and gliotoxic effects of Pb/M. This is in marked contrast to our previous findings that mild hypothermia is protective in the context of a 5 h-long exposure to sevoflurane in infant macaques. These findings demonstrate that brain injury caused by prolonged exposure to Pb/M in the neonatal primate cannot be ameliorated by mild hypothermia., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial infections are common events that significantly impact the clinical course of patients with cirrhosis. As in the general population, infections caused by multi-drug-resistant organisms (MDROs) are progressively increasing in cirrhotic patients, accounting for up to 30-35% of all infections. Nosocomial acquisition and prior exposure to antimicrobial treatment or invasive procedures are well-known risk factors for MDRO infections. Several studies have demonstrated that infections due to MDROs have a poorer prognosis and higher rates of treatment failure, septic shock, and hospital mortality. Due to the increasing rate of antimicrobial resistance, the approach to empirical treatment in cirrhotic patients with life-threatening infections has become significantly more challenging. In order to ensure a prompt administration of effective antibiotic therapy while avoiding unnecessary antibiotic exposure at the same time, it is of utmost importance to choose the correct antimicrobial therapy and administration schedule based on individual clinical characteristics and risk factors and rapidly adopt de-escalation strategies as soon as microbiological data are available. In the present paper, we aimed to provide an overview of the most frequent infections diagnosed in cirrhotic patients, the prevalence and impact of antimicrobial resistance, and potential therapeutic options in this population.

Infants exposed to Zika virus (ZIKV) prenatally may develop birth defects, developmental deficits, or remain asymptomatic. It is unclear why some infants are more affected than others, although enhancement of maternal ZIKV infection via immunity to an antigenically similar virus, dengue virus (DENV), may play a role. We hypothesized that DENV immunity may worsen prenatal ZIKV infection and developmental deficits in offspring. We utilized a translational macaque model to examine how maternal DENV immunity influences ZIKV-exposed infant macaque neurodevelopment in the first month of life. We inoculated eight macaques with prior DENV infection with ZIKV, five macaques with ZIKV, and four macaques with saline. DENV/ZIKV-exposed infants had significantly worse visual orientation skills than ZIKV-exposed infants whose mothers were DENV-naive, with no differences in motor, sensory or state control development. ZIKV infection characteristics and pregnancy outcomes did not individually differ between dams with and without DENV immunity, but when multiple factors were combined in a multivariate model, maternal DENV immunity combined with ZIKV infection characteristics and pregnancy parameters predicted select developmental outcomes. We demonstrate that maternal DENV immunity exacerbates visual orientation and tracking deficits in ZIKV-exposed infant macaques, suggesting that human studies should evaluate how maternal DENV immunity impacts long-term neurodevelopment.

Barbiturates and benzodiazepines are potent GABA A receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life. Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain. Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naïve controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners. These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Use of marmosets in biomedical research has increased dramatically in recent years due, in large part, to their suitability for transgenic applications and utility as models for neuroscience investigations. This increased use includes the establishment of new colonies and involvement of people new to marmoset research. To facilitate the use of the marmoset as a research model, we provide an overview of issues surrounding the ethics and regulations associated with captive marmoset research, including discussion of the history of marmosets in research, current uses of marmosets, ethical considerations related to marmoset use, issues related to importation of animals, and recommendations for regulatory oversight of gene-edited marmosets. To understand the main concerns that oversight bodies have regarding captive biomedical research with marmosets, we developed a brief, 15-question survey that was then sent electronically to academic and biomedical research institutions worldwide that were believed to house colonies of marmosets intended for biomedical research. The survey included general questions regarding the individual respondent's colony, status of research use of the colony and institutional oversight of both the colony itself and the research use of the colony. We received completed surveys from a total of 18 institutions from North America, Europe, and Asia. Overall, there appeared to be no clear difference in regulatory oversight body concerns between countries/regions. One difference that we were able to appreciate was that while biomedical research with marmosets was noted to be either stable or decreasing in Europe, use was clearly increasing elsewhere., (© The Author(s) 2021. Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design., Competing Interests: DHO is a paid consultant for Battelle, devoted to research in the areas of assisting in the design and interpretation of their nonhuman primate ZIKV studies. His relationship does not carry with it any restrictions on publication, and any associated intellectual property will be disclosed and processed according to UW-Madison policy. None of the animals used in this study are involved in any studies with Battelle.

Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement-driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement-blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43-48 hours prior to implantation into ABO-compatible, nonsensitized, MHC-mismatched recipients. Animals were divided into 3 donor-treatment groups: G1 - vehicle, G2 - rhC1INH+heparin, and G3 - heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b-9, and reduced urinary neutrophil gelatinase-associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor-management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD-induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo., Competing Interests: Dr. Lifson is employed by Leidos Biomedical Research, Inc., which exists solely to operate the Frederick National Laboratory for Cancer Research on behalf of the National Institutes of Health and the National Cancer Institute. There are no competing interests to declare.

Zika virus (ZIKV) and dengue virus (DENV) are genetically and antigenically related flaviviruses that now co-circulate in much of the tropical and subtropical world. The rapid emergence of ZIKV in the Americas in 2015 and 2016, and its recent associations with Guillain-Barré syndrome, birth defects, and fetal loss have led to the hypothesis that DENV infection induces cross-reactive antibodies that influence the severity of secondary ZIKV infections. It has also been proposed that pre-existing ZIKV immunity could affect DENV pathogenesis. We examined outcomes of secondary ZIKV infections in three rhesus and fifteen cynomolgus macaques, as well as secondary DENV-2 infections in three additional rhesus macaques up to a year post-primary ZIKV infection. Although cross-binding antibodies were detected prior to secondary infection for all animals and cross-neutralizing antibodies were detected for some animals, previous DENV or ZIKV infection had no apparent effect on the clinical course of heterotypic secondary infections in these animals. All animals had asymptomatic infections and, when compared to controls, did not have significantly perturbed hematological parameters. Rhesus macaques infected with DENV-2 approximately one year after primary ZIKV infection had higher vRNA loads in plasma when compared with serum vRNA loads from ZIKV-naive animals infected with DENV-2, but a differential effect of sample type could not be ruled out. In cynomolgus macaques, the serotype of primary DENV infection did not affect the outcome of secondary ZIKV infection., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: G. Young and H. Dean are employees of Takeda Vaccines, Inc. All other authors declare no conflicts of financial or personal interests.

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8 + T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08 -positive ( Mamu-B*08 + ) RMs. Here we evaluated if robust vaccine-elicited CD8 + T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08 + RMs following mucosal SIV challenges. Ten Mamu-B*08 + RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8 + T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection. IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8 + T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8 + T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection., (Copyright © 2019 American Society for Microbiology.)

Certain major histocompatibility complex class I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. In this study, we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17 Approximately 21% of Mamu-B*17 + and 50% of Mamu-B*08 + RMs control chronic-phase viremia after SIVmac239 infection. Because CD8 + T cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08 + RMs, we investigated whether this might also be true for Mamu-B*17 + RMs. Two groups of Mamu-B*17 + RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (group 1) or together with env (group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the group 1 vaccine regimen had little effect on viral replication compared to group 3, suggesting that unlike Mamu-B*08 + RMs, preexisting SIV-specific CD8 + T cells alone do not facilitate long-term virologic suppression in Mamu-B*17 + RMs. Remarkably, however, 5/8 group 2 vaccinees controlled viremia to <15 viral RNA copies/ml soon after infection. No serological neutralizing activity against SIVmac239 was detected in group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light on the unique mechanism of elite control in Mamu-B*17 + RMs and implicate vaccine-induced, nonneutralizing anti-Env antibodies in the containment of immunodeficiency virus infection. IMPORTANCE A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17 Approximately 21% of Mamu-B*17 + macaques spontaneously controlled chronic phase viremia after SIV infection, an effect that may involve CD8 + T cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17 + macaques with genes encoding immunodominant epitopes in Vif and Nef alone (group 1) or together with env (group 2). Although neither vaccine regimen prevented SIV infection, 5/8 group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in group 1, was associated with vaccine-induced, nonneutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8 + T cells for virologic control in Mamu-B*17 + macaques and implicate anti-Env antibodies in containment of SIV infection., (Copyright © 2018 American Society for Microbiology.)

Here we present a 32-y-old rhesus macaque (Macaca mulatta) with a large recurrent ventral incisional hernia. The initial surgery included midline celiotomy for treatment of endometriosis, in which the animal developed a hernia that was repaired with interposition of mesh. Hernia recurrence at 1 y resulted in a defect measuring 7 × 13 cm, with loss of abdominal domain. Skin breakdown was noted with areas of exposed mesh through the skin with associated acute on chronic infection. Clinically, the animal was lethargic, not eating, and failing to thrive. The present surgical treatment included midline celiotomy, removal of mesh, and attempted primary fascial closure. Due to the large defect and high tension, the fascia could not be closed. To facilitate closure, abdominal component separation technique was used and consisted of skin and subcutaneous dissection, external oblique muscle release, and dissection between the external and internal oblique musculature. This technique allowed for primary fascial closure and resection of excess diseased skin. A piece of polypropylene mesh was placed in a sublay fashion to reinforce the primary fascial closure. The animal tolerated the procedure well and has demonstrated steady weight gain, with no recurrence at 12 mo. Large ventral abdominal hernia defects in after surgery or trauma in NHP can present reconstructive challenges to veterinary surgeons. Failure to achieve a dynamic, low-tension closure can result in hernia recurrence, necessitating additional operations. Abdominal component separation is not commonly used in veterinary surgery and may be a helpful tool in cases of difficult abdominal reconstructions.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a "synthetic swarm" whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.

Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.

Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis-as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses-suggesting that HPgV's protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses-an understudied group of viruses with a high prevalence in the global human population-and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.

Introduction: The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature., Methods: Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed., Results: A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model., Conclusion: We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials.

Zika virus is present in urine, saliva, tears, and breast milk, but the transmission risk associated with these body fluids is currently unknown. Here we evaluate the risk of Zika virus transmission through mucosal contact in rhesus macaques. Application of high-dose Zika virus directly to the tonsils of three rhesus macaques results in detectable plasma viremia in all animals by 2 days post-exposure; virus replication kinetics are similar to those observed in animals infected subcutaneously. Three additional macaques inoculated subcutaneously with Zika virus served as saliva donors to assess the transmission risk from contact with oral secretions from an infected individual. Seven naive animals repeatedly exposed to donor saliva via the conjunctivae, tonsils, or nostrils did not become infected. Our results suggest that there is a risk of Zika virus transmission via the mucosal route, but that the risk posed by oral secretions from individuals with a typical course of Zika virus infection is low.Zika virus (ZIKV) is present in body fluids, including saliva, but transmission risk through mucosal contact is not well known. Here, the authors show that oropharyngeal mucosal infection of macaques with a high ZIKV dose results in viremia, but that transmission risk from saliva of infected animals is low.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFα) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFα upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Background: Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV., Methodology/principal Findings: Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form., Conclusions/significance: An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains., Competing Interests: The authors have declared that no competing interests exist.

Infection with Asian-lineage Zika virus (ZIKV) has been associated with Guillain-Barré syndrome and fetal abnormalities, but the underlying mechanisms remain poorly understood. Animal models of infection are thus urgently needed. Here we show that rhesus macaques are susceptible to infection by an Asian-lineage ZIKV closely related to strains currently circulating in the Americas. Following subcutaneous inoculation, ZIKV RNA is detected in plasma 1 day post infection (d.p.i.) in all animals (N=8, including 2 pregnant animals), and is also present in saliva, urine and cerebrospinal fluid. Non-pregnant and pregnant animals remain viremic for 21 days and for up to at least 57 days, respectively. Neutralizing antibodies are detected by 21 d.p.i. Rechallenge 10 weeks after the initial challenge results in no detectable virus replication, indicating protective immunity against homologous strains. Therefore, Asian-lineage ZIKV infection of rhesus macaques provides a relevant animal model for studying pathogenesis and evaluating potential interventions against human infection, including during pregnancy.

Unlabelled: Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08(+) animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08(+) macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8(+) T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8(+) T-cell response would facilitate the development of elite control in Mamu-B*08(+) animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08(+) animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8(+) T cells. These vaccine-induced effector memory CD8(+) T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8(+) T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08(+) macaques., Importance: Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8(+) T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08(+) rhesus macaques—a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8(+) T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08(+) monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8(+) T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8(+) T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Dengue viruses (DENVs) cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine tetravalent dengue vaccine (TDV) that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3, and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS) is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0) at two different anatomical locations with a needle-free disposable syringe jet injection delivery devices (PharmaJet) in non-human primates. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (2 months later) vaccination schedule. In addition, the vaccine induced CD4(+) and CD8(+) T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3, and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post-challenge). RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas.

Introduction: In thyroid cells, binding of TSH to its receptor increases cAMP levels, sustaining thyrocytes growth and hormone production. The main cAMP effector enzyme is protein kinase A (PKA). Praja2 is a widely expressed RING (Really Interesting New Gene) ligase, which degrades the regulatory subunits of PKA, thus controlling the strength and duration of PKA signaling in response to cAMP. Differentiated thyroid cancer expresses a functional TSH receptor, and its growth and progression are positively regulated by TSH and cAMP signaling., Aim: We aimed to analyze the expression of praja2 in a group of 36 papillary thyroid cancer (PTC), 14 benign nodules, and six anaplastic thyroid cancers (ATC)., Methods: We measured praja2 mRNA levels by quantitative RT-PCR and praja2 expression by Western blot and immunohistochemistry. Possible association between praja2 mRNA and the presence of known mutations was evaluated., Results: We found a statistical significant increase of mRNA levels in PTC tissue samples, compared with benign nodules and ATC. In particular, mRNA levels were maximal in differentiated thyroid cancer (PTC), progressively decreasing in more aggressive tumors, ATC having the lowest amount of praja2 mRNA. Accordingly, higher levels of praja2 protein were detected in lysates from PTC, compared with ATC. By immunohistochemistry, in PTC sections we observed a marked increase of cytoplasmic praja2 signal, which significantly decreased in less differentiated thyroid tumors, completely disappearing in ATC. Studies in cultured cells stably expressing RET/PTC1 oncogene or mutant BRAF revealed a direct correlation between praja2 mRNA levels and malignant phenotype of transformed cells. Similar results were obtained using thyroid cancer tissues carrying the same mutations., Conclusions: praja2 is markedly overexpressed in differentiated thyroid cancer, and its levels inversely correlate with the malignant phenotype of the tumor. Thus, praja2 is a novel cancer-related gene whose expression is linked to the histotype and mutational status of the thyroid tumor.

Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8(+) T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8(+) T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8(+) T-cell response against the Nef RL10 epitope (Nef amino acids 137-146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8(+) T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8(+) T-cell responses can control replication of the AIDS virus.

Introduction: Genomic instability has been proposed to play a role in cancer development and can occur through different mechanisms including telomere association and telomere loss. Studies carried out in our unit have demonstrated that familial papillary thyroid cancer (fPTC) patients display an imbalance, at the germinal level, in telomere-telomerase complex., Aim: We aimed to verify whether familial fPTC patients show an increased spontaneous chromosome fragility., Methods: To this purpose, we compared telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from fPTC patients, unaffected family members, sporadic papillary thyroid cancer patients, and healthy subjects., Results: We demonstrate that fPTC patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared with healthy subjects and sporadic cases in the frame of an otherwise common spontaneous chromosome fragility pattern. A quantitative fluorescence in situ hybridization analysis demonstrates that familial cases display a significant decrease in the telomeric peptide nucleic acid-fluorescence in situ hybridization signal intensity in the metaphase chromosome. Moreover, three copies of the hTERT gene were found only in familial cases, although the result was not statistically significant., Conclusions: These results contribute in defining familial thyroid cancer as a clinical entity characterized by an altered telomere stability, which may be associated with the predisposition to develop the familial form of thyroid cancer.

Introduction: Many studies have found an association between altered telomere length (TL), both attrition or elongation, and cancer phenotype. Recently, we have reported that patients with the familial form of papillary thyroid cancer (FPTC) have short telomeres in blood leucocytes., Aim: To evaluate relative TL (RTL) at somatic level in neoplastic and nonneoplastic tissues of patients with FPTC (n = 30) and sporadic PTC (n = 46)., Methods: RTL was measured by quantitative PCR in neoplastic thyroid tissues, in the corresponding nontumor thyroid tissues (normal contralateral thyroid), and in other extrathyroidal tissues (lymph nodes, muscles, or buccal mucosa). RTL was also measured in adenomas and hyperplastic nodules. In a subset of samples, telomerase expression was measured by quantitative PCR., Results: Mean ± SD RTL of FPTC patients was short in neoplastic thyroid tissues (0.87 ± 0.2) with no difference from the normal contralateral thyroid tissues (0.85 ± 0.11) and extrathyroidal tissues (0.85 ± 0.31). On the contrary, in patients with sporadic PTC, the mean ± SD RTL in the neoplastic tissues (1.73 ± 0.63) was significantly shorter than that found in normal contralateral tissues (2.58 ± 0.89) and extrathyroidal tissues (2.5 ± 0.86). For all tissue samples (cancer, normal thyroid, and nonthyroidal tissues) the mean ± SD RTL of familial cases was shorter (P < 0.0001) than that found in tissues from sporadic PTC. RTL of FPTC was also lower (P < 0.0001) than that of 23 follicular adenomas (1.6 ± 0.7) and 24 hyperplastic nodules (2.2 ± 0.9)., Conclusions: Our results demonstrate that short telomeres are a consistent feature of PTC, which in familial cases, is not restricted to the tumor tissue. This finding suggests that FPTC has a distinct, heritable, genetic background.

Thyroid cancer may have a familial predisposition but a specific germline alteration responsible for the disease has not been discovered yet. We have shown that familial papillary thyroid cancer (FPTC) patients have an imbalance in telomere-telomerase complex with short telomeres and increased telomerase activity. A germline mutation (A339V) in thyroid transcription factor-1 has been described in patients with multinodular goiter and papillary thyroid cancer. In this report, the presence of the A339V mutation and the telomere length has been studied in FPTC patients and unaffected family members. All samples analyzed displayed a pattern typical of the homozygous wild type revealing the absence of the A339V mutation. Shortening of telomeres was confirmed in all patients. We concluded that the A339V mutation in thyroid transcription factor-1 (TITF-1/NKX2.1) is not correlated with the familial form of PTC, even when the tumor was in the context of multinodular goiter.

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8(+) T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8(+) T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8(+) T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4(+) T cells.

Context: Fine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions., Objective: We aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC., Study Design: One hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRgamma mutations. At surgery these nodules were sampled to perform the same molecular testing., Results: Mutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%)., Conclusions: Our findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology.